Biotin supplementation causes erroneous elevations of results in some commercial serum 25-hydroxyvitamin d (25OHD) assays.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes serum samples globally, on a quarterly basis, to assess participants' performance of specific methods for 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25-(OH)2D). DEQAS occasionally circulates samples containing high levels of substances found in certain clinical situations e.g. 25-OHD2, 24,25-(OH)2D3, hypertriglyceridemia. The increased availability and use of health supplements containing biotin has led to case reports of assay interference in methods utilizing a biotin-streptavidin detection system. In October 2018, DEQAS included a serum sample (545) containing exogenous biotin (concentration =586 µg/L) which was analyzed by a total of 683 laboratories using 35 different methods. The same serum sample (544) without exogenous biotin was also included in the 5-sample set. All methods (760 laboratories) performed satisfactorily on sample 544 giving an All-Laboratory Trimmed Mean = 50.2 ± 6.5 nmol/L (±SD, CV = 12.9 %). The target value for this sample 544 (& 555) was 47.4 nmol/L as determined by Centers for Disease Control and Prevention (CDC) Atlanta, Georgia using their LC-MS/MS reference method. In contrast, #545 containing the exogenous biotin was reported by only 683 laboratories and gave an All-Laboratory Trimmed Mean = 66.8 ± 37.6 nmol/L (±SD, CV = 56.3 %). As expected, LC-MS/MS methods (143 labs) reported similar results for both 544 = 48.9 ± 4.4 nmol/L (±SD) and 545 = 48.3 ± 4.5 nmol/L (±SD) showing that assays involving chromatographic steps are unaffected by the presence of biotin. Several of the antibody-based assays including Abbott Architect, DiaSorin Liaison, Beckman Unicel and Siemens Centaur are also unaffected by the addition of biotin. Two assays, IDS-iSYS and Roche Total 25OHD, both of which use biotin-streptavidin, exhibit biotin interference yielding values with a significant positive bias for 545 of 102.6 nmol/L ± 78.7 nmol/L (±SD) and 517.8 nmol/L ± 209.8 nmol/L (±SD) respectively. Interestingly, the failure to report sample 545 data from 77 laboratories is due solely to those running Roche Total 25OHD or Roche Vitamin D Total II assays. Given the prevalence of the adversely affected assays (25 % of DEQAS users) and the high volume of 25OHD testing, clinicians using these assays should, where possible, only measure 25OHD when patients are off biotin.

External Quality Assessment of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) assays.

The discovery that mutations of the CYP24A1 gene are a cause of idiopathic infantile hypercalcemia (IIH) has revived interest in measuring serum 24,25(OH)2D3. Several studies have also suggested that a high 25-hydroxyvitamin D3(25-OHD3):24,25(OH)2D3 ratio might provide additional diagnostic information in the investigation of vitamin D deficiency. Measurement of 24,25(OH)2D3 is necessarily restricted to laboratories with mass spectrometry methods although cross reactivity of the metabolite in immunoassays for 25-OHD is a potential cause of misleading results. The international External Quality Assessment (EQA) scheme for vitamin D metabolites (DEQAS) was set up in 1989. In 2013 DEQAS became an accuracy based EQA for 25-OHD with 'target values' assigned by the National Institute of Standards and Technology (NIST) Reference Measurement Procedure (RMP). A pilot scheme for serum 24,25(OH)2D3 was started in 2015 and participants were asked to measure the metabolite on each of the 5 samples sent out for 25-OHD. Inter-laboratory agreement was poor but this may reflect methodological differences, in particular different approaches to assay standardization. An important potential contribution to reducing variability among assays was the development by NIST of a 24,25(OH)2D3 RMP and its use in assigning values to SRMs 972a, 2973 and 2971, supported by the NIH Office of Dietary Supplements (ODS) as part of the Vitamin D Standardization Program (VDSP) effort.

Quality assessment of vitamin D metabolite assays used by clinical and research laboratories.

The Vitamin D External Quality Assessment Scheme (DEQAS) was launched in 1989 and monitors the performance of 25-hydroxyvitamin D (25-OHD) and 1,25- dihydroxyvitamin D (1,25(OH)2D) assays. In April 2015 a pilot scheme for 24,25-dihydroxyvitamin D (24,25(OH)2D) was introduced. The 25-OHD scheme is accuracy - based with target values assigned by the NIST Reference Measurement Procedure (RMP) for 25-OHD2 and 25-OHD3. A similar method is used to assign values for 3-epi-25-OHD. Five samples of human serum are distributed quarterly to over 1000 participants in 58 countries (April 2016) and clinical laboratories are expected to submit results within approximately 5 weeks. Research laboratories with assays run less frequently are not given a deadline. Archived samples with NIST- assigned values are also available. Performance is assessed on the first four samples with the fifth reserved for investigations e.g. recovery experiments or to assess the influence of other serum constituents such as lipids. DEQAS provides rapid feedback, with an on-line preliminary report available immediately after a participant submits results and a comprehensive report soon after the results deadline. In 2015, DEQAS investigations revealed that several 25-OHD immunoassays under-recovered 25-OHD2 and 25-OHD results were falsely low on a sample with a modestly raised triglyceride concentration. An RMP for 1,25 (OH)2D is not yet available and results are judged against the Method Mean. Free advice is available from the DEQAS Advisory Panel which includes experts on methodology and biostatistics. DEQAS collaborates closely with the Vitamin D Standardization Program (VDSP) and both organizations have successfully worked with participants and manufacturers to improve the accuracy of vitamin D assays.

The development and implementation of quality assurance programs to support nutritional measurements.

The National Institute of Standards and Technology administers quality assurance programs devoted to improving measurements of nutrients and related metabolites in foods, dietary supplements, and serum and plasma samples. These programs have been developed in collaboration with the National Institutes of Health to assist measurement communities in their efforts to achieve accurate results that are comparable among different laboratories and over time. Targeted analytes include micronutrients, botanical markers, nutritional elements, contaminants, fatty acids, and vitamin D metabolites.

Certification of vitamins and carotenoids in SRM 3280 multivitamin/multielement tablets.

A new multivitamin/multielement dietary supplement Standard Reference Material (SRM) has been issued by the National Institute of Standards and Technology (NIST), with certified and reference concentration values for 13 vitamins, 24 elements, and 2 carotenoids. The constituents have been measured by multiple analytical methods with data contributed by NIST and by collaborating laboratories. This effort included the first use of isotope dilution mass spectrometry for value assignment of both fat-soluble vitamins (FSVs) and water-soluble vitamins (WSVs). Excellent agreement was obtained among the methods, with relative expanded uncertainties for the certified concentration values typically ranging from <2% to 15% for vitamins.

Preliminary evaluation of a standard reference material for chiral stationary phases used in liquid and supercritical fluid chromatography.

The applicability of a new Standard Reference Material (SRM) for the evaluation of chiral stationary phase (CSP) performance was demonstrated by utilizing the SRM to characterize the chromatographic behavior of eight commercially available CSPs in liquid and supercritical fluid chromatography. The SRM consists of five ethanolic solutions, each containing one chiral compound. These test mixtures can be used to assess changes in column performance over time and to evaluate lot-to-lot variability in column manufacturing. The SRM was also used to probe the effect of various parameters on column performance.

Chiral selectors from fruit: application of citrus pectins to enantiomer separations in capillary electrophoresis.

Pectins were investigated as chiral selective agents in capillary electrophoresis. Successful enantioresolution of antihistaminic and antimalarial compounds, as well as others, was achieved by utilizing potassium polypectate as the chiral selector. Changes in pH, chiral additive concentration and capillary type were studied in relation to chiral resolution. The effect of degree of esterification of pectin materials on chiral recognition was also evaluated.

Coupled achiral/chiral column techniques in subcritical fluid chromatography for the separation of chiral and nonchiral compounds.

A multicolumn approach was developed to address the limited achiral selectivity of chiral stationary phases. Groups of structurally related compounds, including beta-blockers and 1,4-benzodiazepines, were separated using coupled achiral/chiral stationary phases under subcritical fluid conditions. The achiral selectivity of amino and cyano stationary phases was used to modify the resolution of compounds on a Chiralcel OD chiral stationary phase by combining the achiral and chiral columns in series. In the case of the benzodiazepines, separation of achiral compounds was performed concurrently with the enantioseparation of chiral molecules. The separation of components of a multidrug cough and cold medication was also demonstrated on a cyano column coupled with a Chiralpak AD chiral stationary phase. The use of modified carbon dioxide eluents eliminated the mobile phase incompatibility problems associated with column coupling in liquid chromatography and incorporated the high efficiency of sub- and supercritical fluid chromatography.